Abstract

Nucleic acids-based technology is emerging as a valuable field that integrates research from science and technology to create novel nanodevices and nanostructures with various applicationsin modern nanotechnology. Nowadays, applicationsof RNA-based technologyare employed in biomedical and pharmaceutical research, biosensoring, nanopharmaceutics and others. It has been proven that RNA isa very suitable medium for selfassembly of diverse nanostructures, catalytic nanodevices and cell delivery systems.At the same time, genomics is becoming increasingly valuable for modern medicine due to the advancements made by second generation sequencing technologies. In this review, I discuss various applications of designer ribozymes and diverse RNAbased approaches to medical genomics. The areas discussed include RNA-based approaches for molecular sensoring and diagnostics, antibacterial drug discovery, exogenous control of gene expression, and gene silencing. These approaches havebecomepossible due to the advancement of various methods for engineering functional RNAsas well asthe discoveries made in RNA biology. Furthermore, different RNA-based antisense technologies are reviewed together with methods for nucleic acid delivery to the cell. The research that has been done so far in the field of RNA engineering hasa far-reaching impact on medical genomics, which isthe main focus of this review.

Keywords

Introduction

Nowadays, RNA-based research is emerging as a crossing point
among the natural and engineering sciences. It is an area of exciting
discoveries that includes many different scientific and technological
fields. In fact, many naturalsciences, includingchemistry and biology
along with many types of chemical and biological engineering at a
molecular and sub-molecular level are involved in modern medicine.
The progress achieved by the next generation sequencing (NGS)
technologies [1] in recent years led to the discovery of novel targets
for drug development and diagnostics. The interplay among RNA
engineering, RNA biology, NGS technologies, and medical genomics
creates new possibilities for drug development and molecular
diagnostics. In this review, I presentcurrent and future RNA-based
approaches to medical genomics as the focus set on drug development
[2], molecular diagnostics [3] and forthcoming RNA-based therapeutic
strategies [4].

As RNA-based biotechnology developed, its definition is
leadingmore to the designof functionalsystemsthat can detect and
react to molecular signals from the environment [5]. RNA technology
is a rapidly improving field that involves several major discovers
and technology developments. The discoveries include ribozymes
and riboswitches [6], microRNAs and RNAi [7].The technology
development in general, includes a creation of passive and active
nanostructures, made of many interacting components or integrated
nanodevices. The passive nanostructures are engineered to perform one
particular task, whereas the active structures are designed to execute
several different functions. Active nanostructures can be molecular
sensors, actuators, drug delivery devices, and others. For instance,
molecular sensors can detect the presence or the absence of specific
molecules and pass predefined molecular signals to other sensors. Such
molecular sensors can be used as report systems in many different
applications, including drug discovery through high-throughput
screening arrays. Moreover, molecular sensorscan be engineered to
work as Boolean logic gates [8]. As a result, they can perform logical
operations and solve computational problems [4]. Molecular logic gates can be designed to work together by passing signals among them
in various circuits in vitro as well as in vivo [9].

In fact, RNA-based biotechnology is one of the fastest growing fields
of research based on engineering nanosystems. It is an intersection
between nanotechnology and biology. This paper discusses the main
applications of designer nanostructures and nanodevices based on
RNA molecules. In fact, one of the first nanostructures was made of
RNA using the expertise accumulated in recombinant technology and
molecular biology over the last three decades. RNAhave been proven to
be suitable nanoscale materials. They are relatively easy to synthesize,
amplify, detect, and modify. They can be used both in vitro and in vivo.
Therefore, RNA engineering plays a very important role in modern
nanobiotechnology.

The main advantages of making structures and devices with
RNA are the possibilities of applying many established engineering
methods in conjunction with various tools of molecular biology and
nucleic acid chemistry. In fact, it is easy to chemically synthesize DNA
oligonucleotides and to obtain synthetic RNAs by in vitro transcription
using double-stranded (ds) DNA templates. Moreover, RNAare easy
to amplify, detect, store, and modified. There are powerful software
programs that enable researchers to design automatically specific
RNAmolecules with desired properties.

There are three distinct methods for designing RNA-based
biosensors. They include in vitro selection [10], rational design [11],
and computational methods [8]. RNA sensors have many different
applications in nanobiotechnology. Such applications include molecular computing [12,13], reporter systems for high-throughput
screening assays for antibacterial drug discovery [5,14] synthetic
gene control elements for exogenous regulation [4]. We discuss these
applications in the next sections of this paper.

The initial methods applied for engineering functional nucleic acids
were based on various in vitro selection procedures. As a result, the first
RNA aptamers were obtained. The word “aptamer” comes from the
Latin “aptus” - fit, and Greek “meros” - part. They have the ability to
bind various ligands with high specificity by forming complex threedimensional
structures.The first nucleic acids based aptamers were
obtained by an in vitro selection method called systematic evolution of
ligands by exponential enrichment (SELEX) [15]. In fact, the aptamers
are still produced by various in vitro selection procedures.

These findings suggested that nucleic acids, and particularly RNA,
could be very capable molecular sensors. This makes some researchers
to speculate that RNA molecules may directly sense the presence of
different metabolites in the cell without involvement of any proteins. In
fact, the bid to find such natural RNA aptamersled to the discovery up
to now of 17 different classes of RNA sensors termed riboswitches and
this number is still growing [16]. These newly discovered gene control
elements are usually found in the 5’ untranslated region (UTR) of
messenger (m) RNAs (Figure 1a). They enable mRNAs to adjust their
expression in the presence of specific metabolites [17].

Figure 1: Engineering allosteric ribozymes as reporter systems in HTS arrays
for drug discovery. (A) Supposed secondary structure of the aptamer domain
of the flavin riboswitch in the absence and presence of FMN. (B) The sequence
and the secondary structure of the minimal version of the hammerhead
ribozyme. The arrowhead indicates the cleavage site. (C) The sequence and
the secondary structure of the minimal version of the extended hammerhead
ribozyme from the human parasite Schistosomamansoni, which exhibit highspeed
of cleavage due to its canonical and non-canonical interactions between
the nucleotides of stem II and these of the bulge loop of stem I. (D) Allosteric
ribozymes can be employed as biosensors which recognize small molecules
and cleave external RNA by fusing an aptamer domain (in blue) with a minimal
version of the hammerhead ribozyme. As detection system can be used a
florescence resonance energy transfer (FRET) method. In this method the
external RNA, cleaved by the ribozyme, is attached to a quencher (Q) in its
5’ end and to a reporter (R) in its 3’ end. If the effector is not present, the
allosteric ribozyme is inactive, and the RNA substrate will not be cleaved. (E) If
the effector molecule is present, it binds to the allosteric domain, so that stem
II is formed and the ribozyme will be activated. The activated ribozyme cleaves
the external RNA, which leads to separation of the R from the Q. (F) In this
case, we will detect positive florescence signal. High-throughput machines can
be used to perform this array on microtiter plates.

Genome-wide searches revealed that riboswitches are present
mainly in bacteria. In addition, a few riboswitch classes are also found
to control gene expression in some eukaryote species by alternative
splicing. The genome-wide search of bacterial riboswitches has
important applications to medical genomics. This research results in
the discovery of many riboswitch classes in over 40 different human
pathogenic bacteria [2]. This opens brand new avenues for targeting
new RNAs in antibacterial drug development [18]. The world-wide
demand for new antibiotics is increasing due to the emerging of
many resistant to the common antibiotics stains of human pathogenic
bacteria. Targeting bacterial riboswitches may result in the discovery
of novel classes of antibiotics that have few resistant strains or any for
the time being because there are not yet developed antibiotics specific
targeting bacterial riboswitches.

The riboswitches consist of a ligand-binding (natural aptamer)
domain and an expressionplatform. There are various riboswitchdependent
expression mechanisms, including transcription
termination, prevention of translation, mRNA destabilization, and
others [2]. Riboswitches are classified in different classes according
to their aptamer domains, which have conserved secondary and
tertiary structures. Note that one type of riboswitch can have different
expression platforms. For instance, the FMN riboswitch regulates
gene expression either by transcription termination or by prevention
of translation [19]. In both cases, however, the aptamer domain of
the FMN riboswitch has the some conserved structure that forms
highly specific pocket for FMN. As a result of FMN binding, the flavin
riboswitch alters its structure, which leads to transcription termination
or prevention of translation.

The development of high-throughput screening [20] assays based on
bacterial riboswitches is an essential part in this process. It is important
to create HTS compatible RNA sensors in which the ligand-binding
part of a riboswitch is fused to a reporter domain. Such HTS compatible reporter systems can be obtained by fusing the aptamer domain of a
riboswitch (Figure 1a) with a ribozyme. Ribozymes are conserved RNA
molecules, which possess a catalytic function [21]. For our aim, we can
employ the minimal version of the hammerhead ribozyme (Figure 1b and c). It is called in this way because its specific secondary structure
formed by three stems that looks like a hammerhead [22]. It cleaves
itself at the position indicated by the arrowhead (Figure 1b and c). The
minimal version of the hammerhead ribozyme (Figure 1b) is a short
RNA molecule that requires a high concentration of Mg2+(10 mM) to
exhibit its catalytic function. In contrast, the extended version of the
hammerhead ribozyme (Figure 1c) works at physiologically-relevant
concentration of Mg2+ (1 mM) due to its canonical and non-canonical
interactions between the loop in stem II and the bulge loop in stem I
[23]. Therefore, the minimal version of the hammerhead ribozyme is
used in vitro while the extended form is employed in vivo.

The minimal version of the hammerhead ribozyme can be fused in
stem II with a riboswitch aptamer to obtain different allosteric sensors
(Figure 1d). For this goal, we have used computational methods
based on modeling secondary structures or tertiary interactions in the
presence and in the absence of the ligand. These approachesare very
accurateand time efficient allowing many different riboswitch classes to
be adjusted for HTS assays based on fluorescence detection.We employ
a fluorescence resonance energy transfer (FRET) method for detection
of ribozyme cleavage [14] under multiple turnover conditions. In the
FRET method, two fluorescent dyes are employed. One is attached to
the 5’ end while the other is on the 3’ end. One dye serves as a reporter
(Figure 1e, R) that emits a fluorescence signal upon excitation while
the other one is a quencher (Figure 1e, Q), which absorbs the emitted
signal. However, if the FRET-labeled RNA molecule is cleaved the R
and Q are not anymore in close proximity and the quencher cannot
absorb the emitted by the reporter fluorescence signal.

To make use of such detection system, we have engineered
allosteric hammerhead ribozyme that cleave external FRET-labeled
RNA molecules by opening stems I and III of the ribozyme (Figure 1d and e). When an effector molecule is not present stem II is not formed
and, therefore, the ribozyme is inactive (Figure 1d). In contrary, when
a molecule specifically binds to the aptamer domain stem II of the
ribozyme is formed (Figure 1e). This activates the ribozyme, which
cleaves its substrate RNA (Figure 1e, in red). This ribozyme serves as
a Boolean logic gate with YES function.As a result, the quencher and
reporter are unconnected and the fluorescence emitted by the reporter
can be detected (Figure 1f). This approach can be fully automated in
a HTS format testing hundreds of thousands chemical compound for
specific binding to the aptamer domain of any riboswitch. This can be
used as a first step in riboswitch-based antibacterial drug discovery.
The application of microfluidic technology may further advance this
process [2,24].

RNA-based Circuits

The hammerhead ribozyme can be used to create allosteric
molecular sensors with various Boolean logic function, including NOT,
YES, OR, and AND [8]. In addition, the ribozyme can be designed to
sense not only small molecules but also specific oligonucleotide DNA
and RNA molecules. Moreover, the ribozymes can be programmed to
passsignals from one to other within a molecular circuit. Such molecular
circuits can be used for various applications, including molecular
computing and diagnostics in vitro, and for building synthetic signaling
pathways in vivo. For instance, such a circuit was demonstrated by two
oligonucleotide-sensing ribozymes termed YES-1 and YES-2 (Figure 2). Both ribozymes were designed by a computational procedure [8]
that is a subject for a pending patent application. The computational
procedure for designing oligonucleotide-sensing allosteric ribozymes
is superior to in vitro selection methods both in terms of time efficiency
and design accuracy.

Figure 2: Molecular computing circuits. (A) The secondary structures of two
allosteric ribozymes with YES logic function are depicted. In the absence of an
effector molecule, both ribozymes are inactive. (B) When effector 1 is present,
it binds to the oligonucleotide binding site (OBS1) of the first ribozyme that
activates it. As a result, the YES-1 ribozyme undergoes self-cleavage. (C) The
3’ cleavage fragment (effector 2), released after the self-cleavage of YES-1,
targets the second ribozyme. As a result, YES-2 is also activated.

In the absence of effector oligonucleotides both YES ribozymes
are designed to be in inactive state since stem IV is formed instead of
stem II (Figure 2a). In the presence of oligonucleotide effector-1, the
YES-1 ribozyme is activated since stem II is formed instead of stem
IV when a specific oligonucleotide effector binds the oligonucleotide binding site (OBS) of the ribozyme (Figure 2b). As a result, the YES-1
ribozyme cleaves itself and released a RNA oligonucleotide that serves
as an effector for the YES-2 ribozyme. This results in the activation of
YES-2 ribozyme, which cleaves itself (Figure 2c).

Note that the effector-1 that triggers the circuit can be a DNA
as well as RNA molecule. The ribozymes are very specific. They can
distinguish perfectly well two mismatches over the length of 22 nt.
In addition, several oligonucleotide-sensing allosteric ribozymes can
be designed to work sin parallel. This makes them suitable molecular
sensors for various molecular diagnostic applications.

Integratedrna Based Nanodevices with a Complex Logic
Function as a Tool for Molecular Diagnostics

Integrated nanodevices made of nucleic acidscan combine the function of several molecular logic gates in a single functional
molecule or in an assembly of molecules. Therefore, such devices
can work as integrated circuits at a nanoscale level. There are some
conceptual, designing, and engineering obstacles that need to be
overcomebeforeroutineproduction ofintegrateddevices with nucleic
acids, including RNA.For the time being, there are a few nucleic acids
based devices that integrate the functionality of several logic gates
and can be used for diagnostic purposes. However, even now, there
are examples that provide experimental validation on the feasibility of
engineering such nanodevices.

An example for a RNA-based device that can sense the length of
its target RNA molecules was recently described [3]. This nanodevice
has the complexity of an integrated circuit and can serve as three input
AND Boolean logic gate (Figure 3). Such gate has a true output signal
only in the presence of all three inputs. Our ribozyme-based threeinput
logic gate is capable of sensing the length of its target RNA
molecules. It has two OBSs that can sense two domains of a target RNA
molecule. They are embedded into stem II of the minimal version of the
hammerhead ribozyme. This ribozyme is designed to cleave an external
RNA molecule that has two effector domains and a substrate domain
that are complementary to the OBSs of stem II and the substrate
binding site (SBS) of the ribozyme that forms stem I and III. In fact, if
the spacer between the effector domains and the SBS of the ribozyme
is too short the forms stems I and III will not be formed and the target
RNA will not be cleaved. In contrast, if the spacer of the target RNA
molecule is long enough stems I and III will be properly formed. As a
result the target RNA will be cleaved.

Figure 3: Integrated RNA based nanodevice that sense the length of target
RNAs. (A) The secondary structure of a three input logic gate with AND logic
function is depicted. If the target RNA molecule is too short the stems I and III
are not formed and the ribozyme is inactive. (B) When the target molecule is
long enough all three stems of the ribozyme are formed the substrate RNA is
cleaved.

There are many genetic diseases that are associated with triplet
repeat expansion in mRNAs [25] such as the Huntington’s disorder.
In all these disorders, the mutant mRNA molecules are longer than the
normal ones. The triplet repeats tend to form stable hairpin structures
that makes the length detection of such triplet repeat expansions very
difficult (Figure 3a). To overcome this problem, we can open the hairpin
structure by heating it in the presence of antisense oligonucleotide. As a
result, the hairpin will form a double-strained structure. In this case if
the target mRNA is long enough stems I and III will be formed (Figure
3b). Therefore, the target RNA will be cleaved.This approach can be
used for diagnostic purposes by seeking and destroying specific RNA
molecules with a certain length that are indicative for certain triple
repeat expansion disease.

Allosteric Ribozymes as Designer Cis-Acting Gene
Control Elements

There are many disorders that are associated with abnormal
expression of certain RNA molecules in the cell. We could
oligonucleotide-sensing ribozymes that can detect such abnormal
expression of important RNAs in the cell and to react by altering
expression of exogenous proteins that can overcome the disorder. In
this way, we can achieve two main functions for prevention and curing
various diseases. In the first place, we can monitor the emerging of
the disease indicative RNAs in real time. In the second place, we can
take immediate actions upon detection of such RNAs. This is a future
therapeutic strategy based on allosteric ribozymes that work as cis gene
control elements in the cell.

We can embed high-speed oligonucleotide-sensing ribozymes at
the 3’ end of synthetic mRNAs expression in the cell by viral vectors
(Figure 4). If the designer ribozyme is a NOT gate, it will cleave itself
in the absence of disease indicative RNA in the cell. As a result, the
synthetic mRNA will be decayed and will be not translated into a
protein (Figure 4a). In contrast, in the presence of disease indicative
RNA the ribozyme will be deactivated. The synthetic mRNA will be
translated and therefore, the desired protein will be expression, which
will prevent the disease’s development. For instance, if there are cancer
related RNAs in the cell we could induce the expression of exogenous
p53 that could kill the cancer cell [4].

Figure 4: Ribozyme-mediated regulation of gene expression in eukaryotes.
The gene expression in eukaryotes can be regulated through destabilization of
mRNAs controlled by allosteric ribozymes. We can insert a designer ribozyme
in the 3’ UTR of mRNA that encodes a desired protein. If the ribozyme is
activated, it undergoes self-cleavage that leads to mRNA decay. (A) An
allosteric ribozyme with NOT logical function can be inserted in the 3’ UTR. In
the absence of effector, it cleaves itself, which destabilizes the mRNA. When
a specific ligand is present, the ribozyme is inactivated. The mRNA remains
stable and is translated into protein. (B) When a ribozyme with YES logical
function is inserted in the 3’-UTR, it will be inactive in the absence of effector,
and the gene expression of our protein will be activated. If the effector is
present, it will activate the ribozyme. As a result, the mRNA will be decayed
and there will be not protein synthesis.

We may also sense health indicative RNAs in the cell and expressed
a certain protein only in the absence of such RNAs. To achieve this
we need to employ an allosteric ribozyme with YES logic function as
depicted in (Figure 4b). Note that all high-speed allosteric ribozymes
are based on the extended version of the hammerhead ribozyme
(Figure 1c). In this case, the OBS is introduced in stem III to preserve
the interactions between stems I and II that are responsible the in vivo function of the ribozyme.

Gene Silencing Techniques via Trans-Acting Ribozymes

In modern pharmaceutical research, all tools, which are
manufactured at a nanoscalelevelare termed nanopharmaceutics. There
are two basic types of nanopharmaceutics. One type of them is those
where therapeutic molecules themselves act as drugs. The other type
includes nanocarriers that are used fordrugdelivering. In this case, the
drugs are directly coupled to the nanocarriers. There are various methods
for drug attachment to nanocarriers such as functionalizing, loading,
and encapsulating. As nanocarriers can be used polymerparticles,
nanobeads, nanotubes, and quantumdots. Nanodrugs [26] can be
produced in various forms such as nanosuspension, nanoemulsion,
nanogels, and nanospheres.

It is believed that the nanopharmaceutical research may
be very beneficial for improving drug stability and delivery.
Nanopharmaceutics offer some important advantages such as improved
bioavailability, specific molecular targeting, and controlled release. In
addition, nanopharmaceutics can be engineered to pass intracellular
compartments and the blood to brain barrier andto protect fragile
drugs.They have many different ways of administration including
ocular, pulmonary, oral, topical, parenteral,nasal, and intravenous.

In some cases, the usage of nanopharmaceutics results in reducing
toxicity. However, some nanocarriers have been found to be toxic in
certain ways of administration. This has to be well studied to avoid
adverse effects when applying novel nanopharmaceutics. Nanocarriers
are particulate systems with various sizes between1 and 1000 nm.They
have been successfully used in the treatment of various diseases.
Manytypes of nanocarriers have been introduced up to now. They
include polymermicelle, dendrimers, liposomes, and quantum dots.

RNA molecules can be delivered in the cell by various types of
liposomes. In addition, they can be expressed in the cell by different
types of viral vectors. In fact, nucleic acids show immense potential
to treat cancer, acquired immune deficiency syndrome, neurological
diseases and other incurable human diseases. Intracellular delivery of
nucleic acids is facilitated by nanovectors, both viral and non-viral.A
major advantage of non-viral vectors over viral vectors is safety.
Therefore, RNA molecules can be delivered in many different ways in
the cell.

The advancements of these methods will further empower the
application of various ribozyme-based gene silencing techniques as novel therapeutics.We can employ high-speed allosteric ribozymes
with YES logic function (Figure 5a) for conditional expression of short
hairpin (sh) RNAs only in the presence of some specific RNAs such
as viral RNAs (Figure 5b and c). The shRNAs expressed in the cell
will be cut by the dicer (Figure 5d) and will enter a RNA interference
(RNAi) pathway [27,28]. They can work as microRNAs preventing the
translation of specific mRNAs related to certain disease development
or can decay some target mRNAs in conjunction to the RISC complex
(Figure 5e). Note that these processes tolerate some mismatches and,
therefore, can affect several different mRNAs simultaneity. In fact,
one shRNA sequence usually targets many mRNAs in the cell and
therefore it silences many different genes. That’s why, in most cases,
we cannot afford to constantly expressed shRNAs. Instead we need to
express shRNAs only under certain conditions and for limited time.
Such future approach could deal with this problem. In addition, we can
design a high-speed ribozyme to cleave some specific RNA in the cell
(Figure 5f). However, this approach will be not as efficient as shRNAs
because it will have limited turnovers. All these ribozyme-based gene
silencing techniques may have potent applications to medical genomics
by targeting specific genes in vivo. The future development of RNA
delivery systems in the cell will play a key role in successful applications
of these methods.

Figure 5: Ribozyme-based gene silencing techniques. (A) Without an effector
RNA the YES gate is designed to fold into an inactive structure where a stem
IV is formed instead of stem III. (B) The YES switch binds the effector RNA and
folds into an active state in which stem III is formed. (C) As a result the ribozyme
undergoes self-cleavage and shRNA is produced. (D) Dicer converts shRNA
into dsRNA with sticky ends. (E) In the presence of RISC complex the dsRNA is
single-stranded and used for gene repression either by microRNA function via
translational suppression prevention of RNA translation by RNA decay. When
there is a mismatch(s) with the target mRNA the microRNA function is often
executed. However, mRNA decay is observed not only with perfectly matching
dsRNA but also in the presence of some mismatches. (F) High-speed ribozyme
can be designed to directly cleave a specific RNA in the cell.

Conclusions

RNA basedresearchhas proven to be a productive field of modern
science and technologythat has many important applications to
discovery of novel molecular targets for drug development and
diagnostics, creating reporter systems in HTS arrays, allosteric
biosensors, integrated nanodevices, designer gene control elements,
drug delivery, and others. Moreover, the discovery of new mechanisms
for control of gene expression such as microRNAs, RNAi, and
riboswitches gives us not only better understanding for the complexity
of the RNA function within the cell but also provides us with bigger
opportunities for applying novel RNA-based engineering strategies
both in vitro and in vivo. The advancement of RNA biology and
engineering, on one hand, and the progress achieved in the medical
genomics by the HTS machines, on another hand, offer new exciting
opportunities for developing of novel therapeutic strategies for tackling
various forms of cancer, viral infection diseases, deployment of novel
antibiotics, and others. In addition, RNA engineering promises to
create original gene therapies by reprogramming the cell fate. All this
would be possible if the medical genomics discovers RNAs that are
important targets and/or markers in a disease development process.
Our abilities to identity such RNAs by NGS technologies will improve
over time due to advancement of sequencing technology.

Future Prospective

The application of novel safe and efficient viral expression vectors
will be critical for reaching clinical trials of these methods in the future.
The development of more effective nanoparticle methods for cell
delivery of siRNAs will increase our abilities to develop various RNAi
based therapeutic approaches. There will be some years to come before
the RNA-based technology matured and become a widely spread in
medicine. The proper interaction between the RNA engineering and
medical genomics can significantly speed up this process. The progress
achieved in the computational design of allosteric ribozymes may play
a pivotal role in developing various strategies for disease-dependent
reprograming of cell fate in the near future together with our ability
to specifically inhibit the expression of targeting mRNAs in the cell. In
addition, targeting bacterial riboswitches may lead to the development
of novel classes of antibiotics with less resistance pathogenic bacterial
strains.

Acknowledgement

Robert Penchovsky’s research is supported by a grant number DDVU02/5/2010
awarded by the Bulgarian National Science Fund (NSF).